Anti-idiotypic antibodies to HLA-DR4 and DR2

The aim of our study was to determine whether antibodies recognizing epitopes of HLA-DR antigens (idiotypic antibodies or Ab1) induce the production of anti-idiotypic antibodies (Ab2). We tested the capacity of the F(ab')2 fragment obtained from two sera, one with no anti-HLA antibodies (serum ES) and one depleted by absorption of anti-HLA lymphocytotoxins (serum FH), to block the anti-DR antibodies reacting with the HLA-DR antigens of the immunizing donor. The F(ab')2 fragment obtained from serum ES inhibited the anti-DR2 activity of an earlier post-delivery bleeding obtained from the same woman. The anti-idiotypic antibodies contained by this serum also inhibited the anti-DR2 activity of a reference anti-DR2 antiserum 8W907 and of an anti-MT1 antiserum 8W1231. Similarly, the F(ab')2 fragment obtained from serum FH, after absorption of her anti-DR4 antibody, inhibited the anti-DR4 activity of autologous and homologous antisera. These data suggest that sera of parous women contain anti-idiotypic antibodies directed against regulatory idiotypes of anti-DR antibodies.     

Synthesis of bifunctional antibodies for immunoassays

The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.     

Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli. II. Effects of subunit c-specific polyclonal antibodies.

After incubation of F1-stripped everted membrane vesicles with antibodies against subunit c of the ATP synthase of Escherichia coli the proton translocation through the open F0 channel was blocked. Rebinding of F1 to those vesicles is affected by the antibody concentration used. In general, the use of F(ab')2 or Fab fragments prepared from anti-c antibodies gave similar results. However, using Fab fragments a higher amount of antigenic binding sites was necessary to block the F0 complex completely, whereas extremely low amounts of Fab fragments were necessary to inhibit the binding of F1. This can be explained by an antigenic determinant of subunit c, which is only accessible to the smaller Fab fragments with a molecular mass of approximately 50,000. Incubation of F1-containing everted membranes with anti-c antibodies showed that the binding of the antibodies resulted in a displacement of F1, while simultaneously the proton translocation through F0 has been blocked. Such a displacement can only be observed after incubation with IgG molecules or F(ab')2 fragments. Fab fragments were not able to displace the F1 part, indicating that the ability of antibodies and F(ab')2 fragments to produce cross-links is responsible for the loss of F1 from the membranes.     

Triggering of histamine release from rat mast cells by divalent antibodies against IgE-receptors.

Antibodies against receptor molecules for IgE on rat basophilic leukemic (RBL) cells were prepared by immunization of a rabbit with immune precipitates composed of IgE-receptor complexes and anti-IgE. Antibodies against cell surface components were specifically purified by using RBL cells and rendered specific for mast cells by appropriate absorption. The major antibodies in the final preparation (anti-RBL) were directed against receptor molecules. It was found that the F(ab')2 fragments of anti-RBL induced histamine release from rat mast cells and caused immediate skin reactions in normal rats. These reactions by anti-RBL or its F(ab')2 fragments were inhibited if the receptors on mast cells had been saturated with IgE. The Fab' fragments of anti-RBL could bind with receptors on RBL cells and blocked passive sensitization of mast cells with IgE antibodies, but failed to induce skin reactions and histamine release from normal mast cells. Sensitization of normal rat skin with the Fab' fragment followed by an i.v. injection of anti-rabbit IgG induced skin reactions. The results indicated that bridging of receptor molecules by divalent anti-receptor antibody triggered mast cells for histamine release.     

A human monoclonal IgG1 with anti-idiotypic activity against anti-human thyroglobulin autoantibody

Sixty-one human myeloma proteins (HMP) from patients with multiple myeloma and Waldenstr?m macroglobulinemia were tested for anti-idiotypic (Id) activity against autoantibodies to double-stranded DNA, small nuclear ribonucleoproteins, and human thyroglobulin (HTg), by competitive radioimmunoassays and enzyme immunoassays. An IgG1, lambda HMP from patient BEN with anti-Id activity against antibodies to HTg is reported. IgG1 BEN was not directed toward human Fc fragments and its activity was not related to allotypic determinants. IgG1 BEN molecules recognized Id determinants (idiotopes) on F(ab')2 anti-HTg fragments, but not idiotopes of F(ab')2 fragments of antibodies of other specificities. This observation supports the general significance of Id network interactions in regulation and diversification of immune responses in man.     

Differences of T-cell activation by the anti-CD3 antibodies Leu4 and BMA030

Two anti-CD3 antibodies and their Fab/F(ab')2 fragments were compared with regard to their requirement for secondary signals and generations of intracellular messengers. The anti-CD3 antibody BMA030 was found to require monocyte contact to elicit T-cell mitogenesis. Cross-linking by plastic-bound goat anti-mouse antibodies (panning) failed to activate T cells, even in the presence of recombinant IL-1 or IL-2. In contrast, crosslinking of the anti-CD3 antibody Leu4 or Leu4 fragments was mitogenic in monocyte-free cultures. Measurements of intracellular Ca2+ ([Ca2+]i) and generation of inositol phosphates revealed that binding (+/- panning) of BMA030, Leu4, and their F(ab')2 fragments generated similar amounts of intracellular messengers and thus failed to explain the different responsiveness to passive crosslinking. Since the generation of these messengers was not necessarily followed by proliferation but was always observed when mitogenesis occurred, we conclude that the elevation of [Ca2+]i and the production of inositol phosphates are required but not sufficient to trigger mitogenesis.     

Serum antibodies to human fetal antigens in patients with systemic lupus erythematosus (SLE)

Serum antibodies to human fetal antigens were measured by a radiolabeled anti-immunoglobulin binding assay by using human fetal fibroblasts (Flow cell line No. 1000) as target cells. High titers of IgG antibody to the fetal cells were found in sera of patients with systemic lupus erythematosus (SLE). The antibody reacted with surface membrane antigens shared by various fetal tissues of human and murine origin but not by adult tissues. The reaction of the SLE antibody to the fetal cells was inhibited by heterologous antiserum to the Flow 1000 cells and antiserum to murine embryonic fibroblasts, but not by antiserum to human alpha-fetoprotein or human fibronectin. Absorption of SLE serum with isolated nuclei did not abolish the reaction indicating that these were not anti-nuclear antibodies. The antibody activity was found to reside in the F(ab')2 fragment. The serum titer of the anti-fetal antibody was higher in SLE patients with active disease than those in clinical remission.     

Fluorine-18 labeling of monoclonal antibodies and fragments with preservation of immunoreactivity

A new method is reported for labeling proteins with the positron-emitting nuclide 18F. Initially, 4-[18F]-fluorobenzylamine was prepared in two steps from aqueous [18F]fluoride in high yield. The 18F acylation agent was formed by reaction of this product with disuccinimidyl suberate. Overall yields for the 4-[18F]fluorobenzylamine succinimidyl ester ([18F]SFBS), decay corrected to the end of cyclotron bombardment, were about 30% in a synthesis time of 60 min. After a 15-min reaction, 30-45% (decay corrected) of the [18F]SFBS could be coupled to intact antibodies and their F(ab')2 and Fab fragments. Coupling yields were dependent on protein concentration but not reaction time. HPLC purification of [18F]SFBS was necessary to obtain optimal coupling efficiency and immunoreactivity. The immunoreactivities of 18F-labeled F(ab')2 and Fab fragments of an antimyosin antibody were 89 +/- 5% and 75 +/- 9%, respectively. Biodistribution studies in normal mice demonstrated similar in vivo behavior of 18F-labeled antibody fragments and those labeled with 125I by using N-succinimidyl 3-[125I]iodobenzoate. These results indicate that this method may be useful for labeling monoclonal antibodies and other proteins and peptides with 18F.     

Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.     

Monoclonal anti-idiotypic antibodies to platelet activating factor (PAF) and their interaction with PAF receptors.

Monoclonal anti-idiotypic antibodies (3C3F3E4 and 10D3F8H7) that interact with platelet activating factor (PAF) receptors were generated using an auto-anti-idiotypic approach by immunizing mice with an aldehydic analog of PAF coupled to bovine thyroglobulin. The resulting hybridomas were screened for anti-idiotypic antibody (anti-anti-PAF) with F(ab')2 fragments of affinity-purified polyclonal rabbit anti-PAF antibody. These antibodies displayed internal image properties of PAF and were considered as Ab2 beta according to the following criteria: (a) they bound to F(ab')2 fragments of the affinity-purified rabbit polyclonal anti-PAF antibody that had high affinity for PAF; (b) they inhibited [3H]PAF binding to rabbit polyclonal anti-PAF antibody and its F(ab')2 fragment in a concentration-dependent manner; (c) they displaced [3H]PAF from the anti-PAF antibody/[3H]PAF complex specifically; (d) they inhibited [3H]PAF binding to PAF receptors on rabbit platelet membranes dose dependently; (e) they displaced [3H]PAF from the [3H]PAF/PAF receptor complex specifically; and (f) they stimulated rabbit platelets to aggregate, and this aggregation could be inhibited or totally blocked by specific PAF receptor antagonists WEB 2086 and SRI 63-441. All of the above are consistent with the first successful production of monoclonal antibodies that mimic PAF and interact specifically with the PAF binding domain of PAF receptors on rabbit platelet membranes.     

Asymmetrically glycosylated IgG isolated from non-immune human sera

When human IgG or its F(ab')2 fragment purified from a pool of non-immune sera was passed through a Con A-Sepharose column, 12% of the molecules bound to concanavalin A. While 44% of Fab' and 72% of Fd' fragments obtained from F(ab')2 retained by concanavalin A and eluted with methyl alpha-D-mannoside bound to concanavalin A, the Fab' and Fd' fragments obtained from non-retained F(ab')2 and the L chains and Fc fragments did not interact with the lectin. Only Fd' fragment obtained from the F(ab')2 retained by concanavalin A inhibited the fixation of guinea-pig erythrocytes to concanavalin A. These results are similar to those previously observed for IgG antibodies of different animal species and indicate that partial asymmetric glycosylation is a general phenomenon that is not restricted exclusively to IgG molecules with known specificity.     

Single-step purification of F(ab')2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW.

Hydrophobic interaction high performance liquid chromatography (HPLC) using TSKgel Phenyl-5PW was applicable to single-step purification of F(ab')2 fragments from pepsin digests of mouse monoclonal antibodies of IgG1 class. The digests were applied to the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate. F(ab')2 fragments were adsorbed onto the gel using the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 fragments was homogeneous (purity: higher than 98%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration HPLC. The recovery of the antigen binding site was 42-58%. The cycle time of the Phenyl-5PW HPLC was 45 min, and F(ab')2 of up to 2200 mg was purified in a cycle. This method could be useful especially for large scale purification of F(ab')2 fragments.     

The interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. I. Analysis of the mechanism of binding

We studied the mechanism of binding of radiolabeled, monoclonal anti-H-2 antibodies to mouse spleen cells to determine the number of H-2 antigen molecules per cell. Equilibrium and kinetic data were analyzed in detail according to theoretical models developed for different modes of antibody binding. The results of binding experiments from three monoclonal IgG antibodies (36-7-5, anti-Kk; 27-11-13, anti-DbDd; and 11-4-1, anti-Kk) and their F(ab')2 and F(ab') fragments show that for the IgG and F(ab')2 from all three antibodies, the monovalently and bivalently bound states of the antibody co-exist in rapid equilibrium with one another on the cell surface, with the bivalent state predominating. We show that the relative proportions of the monovalently and bivalently bound species can be estimated from dissociation kinetics experiments, and that once the mode of antibody binding has been established, the density of H-2 determinants on the cell surface can be estimated from equilibrium-binding data. We conclude that the average numbers of H-2K and H-2D molecules on B10.A spleen cells are 5 X 10(4) and 1.1 X 10(5) molecules/cell, respectively.     

Affinity targeting of Sendai virions to desialized human erythrocytes using hybrid antibody molecules

F(ab') fragments obtained from anti-Sendai virus antibodies were chemically coupled to F(ab') fragments obtained from anti-human red blood cell antibodies (anti-hRBC-Ab). This led to the formation of hybrid antibody molecules (anti-SV-anti-hRBC(F(ab')2) each of whose F(ab') fragment possessed different binding specificity. The anti-SV(F(ab'] part of the hybrid molecule interacted specifically with Sendai virus particles, while the anti-hRBC(F(ab'] part interacted with the surface of hRBC. These hybrid antibodies were able to mediate binding and fusion of SV to hRBC, from which the virus receptors were removed by treatment with neuraminidase (desialized hRBC). Neither anti-SV-anti-SV(F(ab')2) nor anti-hRBC-anti-hRBC(F(ab')2) possessed the same ability. Thus, it is shown that soluble, hybrid antibody molecules can effectively mediate functional binding of Sendai virus to virus-receptor-depleted cells.     

CD36-mediated signal transduction in human monocytes by anti-CD36 antibodies but not by anti-thrombospondin antibodies recognizing cell membrane-bound thrombospondin

Mononuclear cells (MNC) treated with anti-CD36 Fab or F(ab')2 fragments and then stimulated with anti-rabbit (F(ab')2 displayed an oxidative burst, suggesting that the crosslinking of CD36 promotes signal transduction in the absence of an Fc receptor involvement. Moreover, intact anti-TSP mediates a weak oxidative burst in MNC, which was strongly enhanced upon pretreatment of monocytes (but not lymphocytes) with TSP. This response, however, was mediated by Fc receptors, not by an involvement of CD36. Other means of crosslinking cell-bound TSP and exposure of MNC to surface-bound TSP failed to promote an oxidative burst. Crosscompetition tests confirmed that the interaction site(s) of TSP with monocytes are distinct from the signal-promoting sites recognized by polyclonal and 3 monoclonal anti-CD36 antibodies.     

Requirements for the opsonic activity of human IgG directed to type 6 group A streptococci: net basic charge and intact Fc region

By two independent techniques for separating human opsonic IgG for group A type 6 streptococci into fast- and slow-migrating fractions, it was found that the opsonic activity was localized within the basic charge population. This charge dependence was found to be a characteristic of the IgG isolated from three individuals. When the fast- and slow-migrating IgG fractions were tested for their ability to bind to purified M6 protein, antibodies in both opsonic and nonopsonic populations exhibited binding activity, with the majority being located within the opsonic IgG in two of the three individuals; the third displayed greater binding in the nonopsonic population. The functional difference observed in the antibody populations to this M antigen may be a reflection of the net charge within the area of the antibody binding site, which suggests that the opsonic antibodies need to bind to acidic residues along the outer surface of the fibrillar M protein molecule. F(ab')2 fragments prepared from both human and rabbit type 6 opsonic IgG were still able to bind to the M6 molecule but were unable to mediate opsonization of type 6 streptococci. However, the F(ab')2 fragments had the capacity to enhance or amplify the opsonic activity of low concentrations of opsonic IgG molecules. The results suggest that the M protein molecule may function as an active inhibitor of phagocytosis and that F(ab')2 fragments from opsonic IgG have the capacity to neutralize the "active" determinants on the molecule, thus allowing lower concentrations of IgG with functional Fc receptors to mediate phagocytosis.     

Triggering of cultured neoplastic mast cells by antibodies to the receptor for IgE.

Cell surface receptors for IgE were isolated from detergent lysates of iodinated, IgE-saturated, rat basophilic leukemia cells by precipitation with anti-IgE antibodies followed by chromatography at acid pH. The isolated material showed a single 125I-band (m.w. approximately 58,000) on gel electrophoresis in sodium dodecyl sulfate and was used to immunize a rabbit. The resulting anti-serum was reacted with lysates of surface iodinated mouse or rat tumor mast cells. Analysis of the precipitates on (10%) gel electrophoresis revealed one major peak comprising greater than 80% of the detectable counts and having an estimated m.w. of approximately 58,000. The antiserum reacted with detergent-solubilized and cell-bound receptors in the presence or absence of excess IgE; it also inhibited the binding of 125I-IgE. Cultured mouse mastocytoma cells never exposed to IgE released 3H-serotonin when incubated with F(ab')2, but not Fab' fragments of the antiserum, which had been rigorously freed of IgE and anti-IgE. The release was inhibited in the presence of excess IgE, was Ca++ dependent, and equaled 80% of the maximum obtained with IgE and anti-IgE. We conclude that aggregation of the receptors for IgE provides the critical signals for cell activation.     

Mitogenic effect on human lymphocytes of insolubilized anti-immunoglobulins. I. Specificity of the stimulating agent

The mitogenic response of peripheral blood lymphocytes to various anti-immunoglobulin reagents has been studied by measuring incorporation of a radioactive thymidine into macromolecules. Coupling of anti-F(ab')2 or anti-light chain antibodies to Sepharose beads leads to a 5-fold increase in their mitogenic capacity with 50-fold less antibodies per culture. Pepsin-digested F(ab')2 fragments had a mitogenic capacity similar to intact antibody molecules. Anti-F(ab')2 antibodies purified by immunoabsorbent columns were found to be more effective as mitogen than unpurified antibody fractions. Antibodies to kappa- or lambda-light chains were found to be mitogenic, whereas antibodies specific to various heavy chain classes failed to induce a significant response. Isolated light chains were much more effective in inhibiting the reaction than isolated mu-chains. It is concluded that insolubilized anti-light chain antibodies are mitogenic to human peripheral blood lymphocytes.     

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants

BACKGROUND: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. MATERIALS AND METHODS: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. RESULTS: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. CONCLUSIONS: Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.